A number of oligonucleotide analogs have been made. One class of oligonucleotides that have been synthesized are the 2'-O-substituted oligonucleotides. Such oligonucleotides have certain unique and useful properties. In U.S. patent application Ser. No. 814,961, filed Dec. 24, 1991, now abandoned, entitled Gapped 2' Modified Phosphorothioate Oligonucleotides, assigned to the same assignee as this application, the entire contents of which are herein incorporated by reference, 2' substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand.
In a recent article, Sproat, B. S., Beijer, B. and Iribarren, A., Nucleic Acids Research, 1990, 18:41, the authors noted further use of 2'-O-methyl substituted oligonucleotides as "valuable antisense probes for studying pre-mRNA splicing and the structure of spliceosomes".
2'-O-methyl and ethyl nucleotides and methods of making the same have been reported by a number of authors.
Robins, M. J., Naik, S. R. and Lee, A. S. K., J. Org. Chem., 39:1891 (1974) reported a low yield synthesis of 2'-O- and 3'-O-methyl guanosine via a stannous chloride catalyzed monomethylation by diazomethane. As was later reported by Robins, M. J., Hansske, F. and Bernier, S. E., Can. J. Chem., 59:3360 (1981), "convenient and high yield methods have been devised for synthesis of the 2'--O-- and 3'-O-methyl ethers of adenosine, cytidine, and uridine . . . However, guanosine has presented significant difficulties." In the foregoing paper, the authors reported an improved synthesis of 2'-O and 3'-O-methyl guanosine. The synthesis was improved by effecting the stannous chloride catalyzed diazomethane methylation of 2,6-diamino-9-(.beta.-D-ribofuranosyl)purine (2-aminoadenosine) in place of guanosine. The diamino purine moiety was then reduced to the corresponding guanine moiety with adenosine deaminase. In a further diazoation reaction described by Singer and Kusmierek, Biochemistry 15:5052 (1976), a mixture of 2' and 3'-O-ethyl guanosine was reported to result from the treatment of guanosine with diazoethane. The alkylation also resulted in alkylation of the heterocyclic base. The alkylated product was treated with base to remove the ethyl group from the heterocyclic base. The resulting product was identified by virtue of having the same UV spectrum as that of guanosine, but a Rf differing from the Rf of guanosine.
A further improvement in the synthesis of 2'-O-methyl nucleosides was reported by Inoue, H., Hayase, Y. Imura, A., Iwai, S., Miura, K. and Ohtsuka, E., Nucleic Acids Research, 15:6131 (1987). This method of synthesis was effected utilizing CH3I in the presence of Ag.sub.2 O. This method proved useful for all of the common nucleotides with the exception of guanosine. As reported by these authors, guanosine proved refractory to this synthetic method. Thus these authors again had to effect the 2'-O-methylation of guanosine with diazomethane. In order to avoid methylation of the amino functionality of the guanine base moiety, the guanine base moiety was blocked with an isobutyryl group. Additionally, to avoid methyl esterification of the 3'-O functionality of the sugar moiety, a TIPDS (tetraisopropyldisiloxane) blocking group was used to block both the 3' and the 5' hydroxyls of the sugar moiety.
Sproat et al., supra and Sproat, B. S., Iribarren, A. M., Garcia, R. G. and Beijer, B., Nucleic Acids Research, 19:733 (1991) addressed the synthesis of 2'-O-methyl guanosine (and 2'-O-allyl guanosine). In both of these Sproat et al. publications, the investigators presented a further synthetic pathway to 2'-O-methylguanosine and 2'-O-allylguanosine. They characterized the further pathway with respect to the prior known synthetic methods as "avoids(ing) . . . the use of the highly toxic and potentially explosive reagent diazomethane" and being "far superior to the use of silver oxide/methyl iodide." This same synthetic method of the Sproat et al. investigators is also published in B. S. Sproat and A. I. Lamond, "2'-O-Methyloligoribonucleotides: synthesis and applications," Oligonucleotides and Analogues, ed. F. Eckstein, (IRL Press, 1991) which described syntheses of 2'-O-methylribonucleoside-3'-O-phosphoramidites. The uridine phosphoramidite synthesis described therein requires both base and sugar protection of the starting nucleoside prior to alkylation. Only after the base and sugar protecting groups are in place on the uridine is it then alkylated. Post alkylation, the base protecting group is removed followed by 5'-O-dimethoxytritylation and phosphitylation. The cytidine phosphoramidite synthesis described by Sproat and Lamond utilizes (and thus requires) the base and sugar blocked 2'-O-methyl uridine analog. This analog is then converted to a blocked cytidine analog, the blocking group is removed from the sugar, the analog is dimethoxytritylated and finally phosphitylated. The guanosine phosphoramidite synthesis taught by Sproat and Lamond starts from a 2-amino-6-chloronucleoside having 3' and 5' sugar hydroxy groups blocked. This nucleoside is converted to a 2,6-dichloro derivative. The dichloro compound is then 2'-O-alkylated. Following O-alkylation, the dichloro compound is converted to a diazido intermediate. The diazido intermediate is in turn converted to a diamino intermediate. The diamino intermediate is then deaminated to the guanosine analogue. The 2-amino group of the guanosine analogue is blocked followed by dimethoxytritylation and finally phosphitylation. This guanosine procedure is also published in Sproat, et. al., Nucleic Acids Research, 1991 19:733.
The above synthetic procedures involve multiple steps and numerous reagent treatments--9 different reagent treatments for uridine, 10 for cytidine and 12 for guanosine. For the cytidine and guanosine compounds at least one of the reagents that is required is not readily available and thus is a very expensive reagent.
Other groups have taught the preparation of other 2'-O-alkylated nucleosides. 2'-O-methylthiomethylguanosine, was reported by Hansske, F., Madej, D. and Robins, M. J., Tetrahedron, 40:125 (1984). It was produced as a minor by-product of an oxidization step during the conversion of guanosine to 9-.beta.-D-arabinofuranosylguanine, i.e. the arabino analogue of guanosine. The addition of the 2'-O-methylthiomethyl moiety is an artifact from the DMSO solvent utilized during the oxidization procedure. The 2'-O-methylthiomethyl derivative of 2,6-diaminopurine riboside was also reported in the Hansske et al. publication. It was also obtained as an artifact from the DMSO solvent.
In addition, Gladkaya, et al., Khim. Prir. Soedin., 1989, 4, 568 discloses N.sub.1 -methyl-2'-O-(tetrahydropyran-2-yl) and 2'-O-methyl guanosine. Sproat, et al., Nucleic Acids Research, 1991, 19, 733 teaches the preparation of 2'-O-allyl-guanosine. Allylation of guanosine required a further synthetic pathway. Iribarren, et al., Proc. Natl. Acad. Sci., 1990, 87, 7747 also studied 2'-O-allyl oligoribonucleotides. Iribarren, et al. incorporated 2'-O-methyl-, 2'-O-allyl-, and 2'-O-dimethylallyl-substituted nucleotides into oligoribonucleotides to study the effect of these RNA analogues on antisense analysis. Iribarren found that 2'-O-allyl containing oligoribonucleotides are resistant to digestion by either RNA or DNA specific nucleases and slightly more resistant to nucleases with dual RNA/DNA specificity, than 2'-O-methyl oligoribonucleotides. However, Iribarren found that 2'-O-dimethylallyl containing oligoribonucleotides exhibited reduced hybridization to complementary RNA sequences as compared to 2'-O-methyl oligoribonucleotides. Thus, Iribarren suggested that further attempts to prepare alkylated RNA probes, especially those superior to 2'-allyl cytidine containing oligoribonucleotides should be limited to 2'-O-alkyl groups containing less than five carbon atoms.
Certain oligonucleotides containing 2'-O-alkyl substituted nucleotides are promising candidates for use as human pharmaceuticals. Those having long chain alkyl groups (i.e. four or more carbon atoms) are particularly useful. For example, long chain alkyl groups may accomodate functional groups in appropriate orientation with the opposing strand upon strand hybridization. Thus 2'-O-long chain alkyl nucleotides such as 2'-O-long chain alkyl guanosine nucleotides are highly desireable in some cases. For use in large scale therapeutic testing and eventually for human pharmaceutical use, large amounts of these oligonucleotides must be synthesized. The large amounts of oligonucleotides in turn requires large amounts of the 2'-O-alkyl nucleoside phosphoramidites used in synthesizing the oligonucleotides. Consideration must therefore be given to both cost and purity of the starting phosphoramidites used in the synthesis of such oligonucleotides. As a general premise, as the number of synthetic steps increases, the cost of manufacture increases. Further as the number of steps increases, quality control problems escalate. In view of this, it is evident that there is a great need for new and improved procedures for preparing nucleosides and nucleoside phosphoramidites.